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WO1996033209A1 - Derives peptidyle utilises comme inhibiteurs de cysteine proteinases pro-apoptotiques - Google Patents

Derives peptidyle utilises comme inhibiteurs de cysteine proteinases pro-apoptotiques Download PDF

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Publication number
WO1996033209A1
WO1996033209A1 PCT/CA1996/000247 CA9600247W WO9633209A1 WO 1996033209 A1 WO1996033209 A1 WO 1996033209A1 CA 9600247 W CA9600247 W CA 9600247W WO 9633209 A1 WO9633209 A1 WO 9633209A1
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Prior art keywords
amino
apopain
substituted
hydroxy
aryl
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PCT/CA1996/000247
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English (en)
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Michel Gallant
Marc Labelle
Yves Gareau
Donald W. Nicholson
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Merck Frosst Canada Inc.
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Priority to AU53290/96A priority Critical patent/AU5329096A/en
Publication of WO1996033209A1 publication Critical patent/WO1996033209A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C12N9/6475—Interleukin 1-beta convertase-like enzymes (3.4.22.10; 3.4.22.36; 3.4.22.63)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • this invention is directed to pro-apoptotic cysteine proteinases, such as apopain, the DNA encoding for same or its anti-sense, and an assay for identifying agents that modulate these proteinases.
  • this invention is directed to peptidyl derivatives which are modulators of the activity of pro-apoptotic cysteine proteinases, such as apopain, and their use in therapy and in identifying agents useful in the treatment of pro-apoptotic cysteine proteinase-mediated diseases.
  • Apoptosis constitutes a systematic means of cell suicide within an organism during normal morphogenesis, tissue remodelling as well as in response to pathogenic infections or other irreparable cell damage.
  • Inappropriate apoptosis may underlie the aetiology of human diseases such as Alzheimer's, Parkinson's and Huntington's diseases, immune deficiency and autoimmune disorders, ischemic cardiovascular and neurological injury, alopecia, leukemias, lymphomas and other cancers, which therefore makes the control of apoptosis an important potential target for therapeutic intervention 1 -4.
  • human diseases such as Alzheimer's, Parkinson's and Huntington's diseases, immune deficiency and autoimmune disorders, ischemic cardiovascular and neurological injury, alopecia, leukemias, lymphomas and other cancers, which therefore makes the control of apoptosis an important potential target for therapeutic intervention 1 -4.
  • PARP poly(ADP-ribose) polymerase
  • PARP is proteolytically cleaved at the onset of apoptosis by a hitherto- unidentified protease with properties that resemble those of ICEl8,19.
  • the cleavage site within PARP (DEVD216-G217) resembles one of the two sites in proIL-l ⁇ (FEAD27-G28) that are recognized and cleaved by ICE.
  • Proteolytic cleavage of PARP at this site results in the separation of the two zinc-finger DNA-binding motifs in the amino- terminus of PARP from the automodification and poly(ADP- ribos)ylating catalytic domains located in the carboxy-terminus of the polypeptide.
  • This cleavage precludes the catalytic domain of PARP from being recruited to sites of DNA damage and presumably disables the ability of PARP to coordinate subsequent repair and genome maintenance events.
  • the Ca2+/Mg2+-dependent endonuclease implicated in the internucleosomal DNA cleavage that is a hallmark of apoptosis is negatively regulated by poly(ADP- ribos)ylation20-22. Loss of normal PARP function would therefore render this nuclease highly activated in dying cells.
  • Interleukin-l ⁇ is a major mediator of chronic and acute inflammation. It is synthesized as an inactive 31 kDa precursor (pIL-l ⁇ ) that is processed to its mature 17.5 kDa form (mJL-l ⁇ ) by interleukin-l ⁇ converting enzyme (ICE), a cysteine proteinase.
  • ICE interleukin-l ⁇ converting enzyme
  • ICE-like genes including the namatode cell death abnormal gene (CED-3) of Caenorhabiditis elegans, Caenorhabiditis briggsae and Caenorhabiditis vulgaris, the murine neuronal precursor cell gmbroyonic ⁇ evelopmentally downregulated (NEDD-2) gene and its human homologue ICH-1, as well as CPP32 which was cloned from Jurkat cells.
  • NEDD-2 murine neuronal precursor cell gmbroyonic ⁇ evelopmentally downregulated
  • ICH-1 human homologue
  • CPP32 murine neuronal precursor cell gmbroyonic ⁇ evelopmentally downregulated
  • ICErel-IH interleukin-l ⁇ converting enzyme - related cysteine proteinase III; U.S.S.N. 08/224,930, filed April 8, 1994.
  • sequence identities of ICE re l-II and ICErel-IH with ICE are 61% and 56% respectively. All known sequences for ICE, CED-3 and other members of this new family of cysteine proteinases contain the pentapeptide sequence -Gln-Ala-Cys-Arg-Gly- surrounding the catalytic cysteine of ICE or its equivalent in the other members.
  • the five known members of the ICE CED-3 family of cysteine proteases which are of human origin are each capable of initiating an apoptotic response when transfected into host cells; however, it is possible that overexpression of any protease may cause non-specific induction of cell death. Cytoplasmic expression of other proteases, such as trypsin, chymotrypsin, proteinase K or granzyme B, for example, have also been shown to induce apoptosis27,28.
  • apopain an active form of CPP32 , designated apopain, is the enzyme responsible for the specific proteolytic breakdown of PARP that occurs at the onset of apoptosis. Furthermore, we show that inhibition of apopain-mediated PARP cleavage attenuates apoptosis in vitro, demonstrating the central role played by this protease in the apoptosis of mammalian cells.
  • the term 'apopain' is used herein to describe the enzymatically active form of the pro-apoptotic cyteine protease responsible for cleavage of poly(ADP-ribose) polymerase.
  • the invention describes for the first time the identification of this enzyme and it is thus named apopain in accordance with I.U.B. nomenclature guidelines using the prefix 'apop' to indicate its role in apoptosis and the suffix 'ain' which is preferred by the I.U.B. for naming all cysteine proteases.
  • CPP32 cyste protease protein of 32 kDa
  • CPP32 cyste protease protein of 32 kDa
  • the present invention is directed to an isolated and purified enzyme designated apopain, methods of using apopain to screen for compounds which modulate the activity of apopain, and compounds identifed by the screens.
  • a synthetic DNA molecule encoding full length apopain is prepared based on the primary amino acid sequence of the purified enzyme.
  • the synthetic apopain-encoding DNA is formulated so as to optimize expression in a variety of recombinant hosts.
  • the DNA clones produce recombinant full-length apopain and derivatives thereof.
  • Purified native apopain and recombinant apopain are useful for identifying modulators of apopain activity and hence modifiers of pathological conditions related to the pro-inflammatory or pro-apoptotic effects of apopain.
  • Apopain antisense molecules are useful for therapeutically reducing or eliminating the pro-inflammatory or pro-apoptotic effects of apopain, whereas gene transplantation or gene therapy with apopain is useful for enhancing the pro-inflammatory or pro-apoptotic effects of apopain.
  • this invention relates to substituted peptidyl derivatives of formula I
  • this invention relates to inhibitors of the pro-apoptotic proteolytic activity of thiol proteinases which cause apoptosis at least in part by disabling the normal biological function of poly(ADP-ribose)polymerase.
  • Figure 1 shows PARP cleavage activity in spontaneously apoptotic osteosarcoma cells.
  • Figure 2 shows inhibition of PARP cleavage in apoptotic osteosarcoma cell extracts.
  • Figure 3 shows purification of the PARP cleavage protease from THP-1 cells.
  • Figure 4 shows the structure of PARP cleavage protease; apopain, which is derived from the inactive proenzyme CPP32.
  • Figure 5 shows the kinetic analysis of apopain and a potent inhibitor using a fluorogenic substrate.
  • Figure 6 shows in vitro apoptosis and selective inhibition by
  • the present invention is directed to an isolated and purified enzyme designated apopain, methods of using apopain to screen for compounds which modulate the activity of apopain, and compounds identifed by the screens.
  • a synthetic DNA molecule encoding full length apopain is prepared based on the primary amino acid sequence of the purified enzyme.
  • the synthetic apopain-encoding DNA is formulated so as to optimize expression in a variety of recombinant hosts.
  • the DNA clones produce recombinant f ill-length apopain and derivatives thereof.
  • Purified native apopain and recombinant apopain are useful for identifying modulators of apopain activity and hence modifiers of pathological conditions related to the pro-inflammatory or pro-apoptotic effects of apopain.
  • Apopain antisense molecules are useful for therapeutically reducing or eliminating the pro-mflammatory or pro-apoptotic effects of apopain, whereas gene transplantation or gene therapy with apopain is useful for enhancing the pro-inflammatory or pro-apoptotic effects of apopain.
  • These therapies are beneficial in the treatment of immune, proliferative and degenerative diseases including, but not limited to, immune deficiency syndromes (such as AIDS), autoimmune diseases, pathogenic infections, cardiovascular and neurological injury, alopecia, aging, cancer, Parkinson's disease and Alzheimer's disease.
  • immune deficiency syndromes such as AIDS
  • autoimmune diseases pathogenic infections
  • cardiovascular and neurological injury such as alopecia
  • alopecia aging
  • cancer Parkinson's disease
  • Parkinson's disease Alzheimer's disease.
  • Apoptosis constitutes a systematic means of cell suicide within an organism during normal morphogenesis, tissue remodelling as well as in response to pathogenic infections or other irreparable cell damage. Inappropriate apoptosis may underlie the aetiology of human diseases such as Alzheimer's, Parkinson's and Huntington's diseases, immune deficiency and autoimmune disorders, ischemic cardiovascular and neurological injury, alopecia, leukemias, lymphomas and other cancers, which therefore makes the control of apoptosis an important potential target for therapeutic intervention 1-4.
  • Several of the biochemical events that contribute to apoptotic cell death have recently been elucidated.
  • ced-3 encodes a putative cysteine protease which is related to mammalian interleukin-l ⁇ converting enzyme (ICE)6, the first identified member of a new family of cysteine proteases with the distinguishing feature of a near absolute specificity for aspartic acid in the Si subsite7,8.
  • ICE mammalian interleukin-l ⁇ converting enzyme
  • Deletion or mutation of the ced-3 gene completely prevented the apoptotic death of all cells that were otherwise destined to die, and both CED-3 as well as ICE induced apoptosis when transfected into a variety of host cells6,9,10.
  • CED-3 could be prevented by co-transfection with the nematode death suppressor gene ced-9 and to some degree by its mammalian counterpart, the proto-oncogene bcl-2.
  • the fate of eucaryotic cells may therefore reside in the balance between the opposing pro-apoptotic effects of an ICE/CED-3-like protease and an upstream regulatory mechanism involving Bcl-2 and/or its homologues.
  • PARP poly(ADP-ribose) polymerase
  • PARP is proteolytically cleaved at the onset of apoptosis by a hitherto- unidentified protease with properties that resemble those of ICE 8,19.
  • the cleavage site within PARP (DEVD216-G217) resembles one of the two sites in proIL-l ⁇ (FEAD27-G28) that are recognized and cleaved by ICE.
  • Proteolytic cleavage of PARP at this site results in the separation of the two zinc-finger DNA-binding motifs in the amino- terminus of PARP from the automodification and poly(ADP- ribos)ylating catalytic domains located in the carboxy-terminus of the polypeptide. This cleavage precludes the catalytic domain of PARP from being recruited to sites of DNA damage and presumably disables The ability of PARP to coordinate subsequent repair and genome maintenance events. Furthermore, the Ca2+/Mg2+-dependent endonuclease implicated in the internucleosomal DNA cleavage that is a hallmark of apoptosis is negatively regulated by poly(ADP- ribos)ylation20-22. Loss of normal PARP function would therefore render this nuclease highly activated in dying cells.
  • the five known members of the ICE CED-3 family of cysteine proteases which are of human origin are ICE, ICErel-H, ICErel- ⁇ i, ICH-1 and CPP3223-26. Each is capable of initiating an apoptotic response when transfected into host cells; however, it is possible that overexpression of any protease may cause non-specific induction of cell death. Cytoplasmic expression of other proteases, such as trypsin, chymotrypsin, proteinase K or granzyme B, for example, have also been shown to induce apoptosis27,28.
  • ced-3 In the nematode C. elegans, deletion or mutation of a single gene, ced-3, abolishes apoptotic death5.
  • ced-3 was found to be homologous to the gene for mammalian interleukin-l ⁇ converting enzyme (ICE)6, which encodes a protease whose only known function is the cleavage of the inactive 31 kDa proIL-l ⁇ cytokine precursor to the active 17 kDa form.
  • ICE mammalian interleukin-l ⁇ converting enzyme
  • prICE is in fact apopain CPP32 and that apopain/CPP32 is the specific ICE/CED-3-like cysteine protease that cleaves PARP in mammalian cells.
  • the central role played by apopain CPP32 in mammalian cell death is further substantiated by potent and selective inhibitors which prevent apoptosis from occurring in vitro.
  • the pharmacological modulation of apopain activity may therefore be an appropriate point for therapeutic intervention in pathological conditions where inappropriate apoptosis is prominent.
  • the cloned apopain cDNA may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant apopain.
  • Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.
  • An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
  • a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
  • a strong promoter is one which causes mRNAs to be initiated at high frequency.
  • Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
  • mammalian expression vectors may be used to express recombinant apopain in mammalian cells.
  • Commercially-available mammalian expression vectors which may be suitable for recombinant apopain expression, include but are not limited to, pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV- MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).
  • DNA encoding apopain may also be cloned into an expression vector for expression in a recombinant host cell.
  • Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria, yeast, mammalian cells and insect cells.
  • Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO- Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • CV-1 ATCC CCL 70
  • COS-1 ATCC CRL 1650
  • COS-7 ATCC CRL 1651
  • CHO- Kl ATCC CCL 61
  • 3T3 ATCC CCL 92
  • NIH/3T3 ATCC CRL 1658
  • HeLa ATCC CCL 2
  • C127I ATCC CRL 1616
  • BS-C-1 ATCC CCL 26
  • MRC-5 ATCC CCL 171.
  • the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, infection, protoplast fusion, and electroporation.
  • the expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce apopain protein. Identification of apopain expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-apopain antibodies, and the presence of host cell-associated apopain activity.
  • Expression of apopain cDNA may also be performed using in vitro produced synthetic mRNA.
  • Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes.
  • apopain cDNA sequence(s) that yields optimal levels of enzymatic activity and/or apopain protein
  • modifed apopain cDNA molecules are constructed.
  • Host cells are transformed with the cDNA molecules and the levels of apopain RNA and protein are measured.
  • apopain protein in host cells are quantitated by a variety of methods such as immunoaffinity and/or ligand affinity techniques, apopain-specific affinity beads or apopain-specific antibodies are used to isolate 35s-methionine labelled or unlabelled apopain protein.
  • Labelled apopain protein is analyzed by SDS-PAGE.
  • Unlabelled apopain protein is detected by Western blotting, ELISA or RIA assays employing apopain specific antibodies.
  • apopain protein may be recovered to provide apopain in active form.
  • Recombinant apopain may be purified from cell lysates or from conditioned culture media, by various combinations of, or individual application of fractionation, or chromatography steps that are known in the art.
  • recombinant apopain can be separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or polyclonal antibodies specific for full length nascent apopainor polypeptide fragments of apopain
  • the recombinant protein may be used to generate antibodies.
  • antibody as used herein includes both polyclonal and monoclonal antibodies, as well as fragments thereof, such as, Fv, Fab and F(ab)2 fragments that are capable of binding antigen or hapten.
  • Monospecific antibodies to apopain are purified from mammalian antisera containing antibodies reactive against apopain or are prepared as monoclonal antibodies reactive with apopain using standard techniques.
  • Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for apopain.
  • Homogenous binding refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the apopain, as described above.
  • Enzyme-specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of apopain either with or without an immune adjuvant.
  • Monoclonal antibodies (mAb) reactive with apopain may be prepared by conventional methods, such as by immunizing inbred mice with apopain.
  • the mice are immunized with about 0.1 mg to about 10 mg, preferably about 1 mg, of apopain in about 0.5 ml buffer or saline inco ⁇ orated in an equal volume of an acceptable adjuvant. Freund's complete adjuvant is preferred.
  • the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 0.1 to about 10 mg of apopain in a buffer solution such as phosphate buffered saline (PBS) by the intravenous (IV) route.
  • PBS phosphate buffered saline
  • Lymphocytes from antibody-positive mice are obtained by removing spleens from immunized mice by standard procedures known in the art.
  • Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner under conditions which will allow the formation of stable hybridomas.
  • Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.
  • DMEM Dulbecco's Modified Eagles Medium
  • Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using apopain as the antigen.
  • SPIRA solid phase immunoradioassay
  • the culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb.
  • Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.
  • In vitro production of anti-apopain is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb.
  • the mAb are purified by techniques known in the art.
  • Antibody liters of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RLA) techniques. Similar assays are used to detect the presence of apopain in body fluids or tissue and cell extracts. Methods such as those described above may be used to produce monospecific antibodies may be utilized to produce antibodies specific for apopain polypeptide fragments or full-length nascent apopain polypeptide.
  • serological or immunological assays include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RLA) techniques. Similar assays are used to detect the presence of apopain in body fluids or tissue and cell extracts. Methods such as those described above may be used to produce monospecific antibodies may be
  • Apopain antibody affinity columns are made by adding the antibodies to a gel support, such as Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support.
  • the antibodies are then coupled to the gel via amide bonds with the spacer arm.
  • the remaining activated esters are then quenched with 1M ethanolamine HCI (pH 8).
  • the column is washed with water followed by 0.23 M glycine HCI (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
  • kits containing apopain cDNA, antibodies to apopain or apopain protein may be prepared. Such kits are used to detect DNA which hybridizes to apopain DNA or to detect the presence of apopain protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses and epidemiological studies.
  • the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of apopain DNA, apopain RNA or apopain protein.
  • the recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of apopain.
  • a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container.
  • the carrier would further comprise reagents such as recombinant apopain protein or anti-apopain antibodies suitable for detecting apopain.
  • the carrier may also contain means for detection such as labeled antigen or enzyme substrates or the like.
  • Nucleotide sequences that are complementary to the apopain encoding cDNA sequence can be synthesized for antisense therapy.
  • These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-0-alkyLRNA, or other apopain antisense oligonucleotide mimetics.
  • Apopain antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harbouring the antisense sequence, apopain antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce apopain activity.
  • Apopain gene therapy may be used to introduce apopain into the cells of target organs.
  • the apopain gene can be ligated into viral vectors which mediate transfer of the apopain DNA by infection of recipient host cells.
  • Suitable viral vectors include retro virus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like.
  • apopain DNA can be transferred into cells for gene therapy by non-viral techniques including receptor- mediated targetted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations of them are suitable for ex vivo as well as in vivo apopain gene therapy, apopain gene therapy may be particularly useful for the treatment of diseases where it is beneficial to elevate apopain activity.
  • compositions comprising apopain DNA or apopain protein may be formulated as described elsewhere in this application or according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein or DNA.
  • compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose apopain related disorders.
  • the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
  • the pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
  • this invention is also directed to those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid.
  • a sequence bearing one or more replaced codons will be defined as a degenerate variation.
  • mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
  • DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide.
  • Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate.
  • a “functional derivative” of apopain is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of apopain.
  • the term “functional derivatives” is intended to include the “fragments,” “variants,” “degenerate variants,” “analogs” and “homologs” or to “chemical derivatives” of apopain.
  • fragment is meant to refer to any polypeptide subset of apopain.
  • variant is meant to refer to a molecule substantially similar in structure and function to either the entire apopain molecule or to a fragment thereof.
  • a molecule is "substantially similar" to apopain if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
  • analog refers to a molecule substantially similar in function to either the entire apopain molecule or to a fragment thereof.
  • the term "chemical derivative” describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solublity, half-life, absorption, etc of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
  • the present invention is also directed to methods for screening for compounds which modulate that expression of DNA or RNA encoding apopain as well as the function of apopain protein in vivo. Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
  • Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding apopain or the function of apopain protein.
  • Compounds that modulate the expression of DNA or RNA encoding apopain or the function of apopain protein may be detected by a variety of assays.
  • the assay may be a simple "yes/no" assay to determine whether there is a change in expression or function.
  • the assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
  • the invention encompasses compounds of formula I.
  • aryl Cl-6 alkyl wherein the aryl group is selected from the group consisting of:
  • aryl wherein the aryl is selected from the group consisting of phenyl, 1-napthyl, 2-naphthyl, 9-anthracyl and 2, 3, or 4 pyridyl, and mono-, di or tri-substituted derivatives thereof, wherein the substituents are individually selected from the group consisting of
  • AAI is independently selected from the group consisting of (a) a single bond, and
  • AA2 is an amino acid of formula All
  • AA3 is an amino acid of formula .Ai ⁇
  • R7, R8 and R9 are each independently selected from the group consisting of
  • aryl Cl-6 alkyl wherein aryl is phenyl, 1- or 2-naphthyl, 9-authracyl, or 2-, 3- or 4- pyridyl, and wherein the aryl may be mono and di-substituted, the substituents being each independently Cl-6alkyl, halo, hydroxy, Ci-6alkyl amino, Ci-6alkoxy, Cl-6alkylthio, and Ci-6alkylcarbonyl.
  • aryl Cl-6 alkyl wherein the aryl group is selected from the group consisting of:
  • R2 is (a) hydrogen, OH, Cl-6 alkyloxy or Cl-6 perfluoroalkyl; R7, R8 and R ⁇ are each independently selected from the group consisting of (a) hydrogen,
  • aryl is as defined previously, and wherein the aryl may be mono and di-substituted, the substituents being each independently Ci-6alkyl, halo, hydroxy, Cl-6alkyl amino, Cl-6alkoxy, Ci-6alkylthio, and Cl-6alkylcarbonyl.
  • AAl, AA2 and -AA3 are each independently selected from the group consisting of the L- and D- forms of the amino acids including glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxy lysine, histidine, arginine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, ornithine, b- alanine, homoserine, homotyrosine, homophenylalanine and citrulline.
  • Rl is Cl-3alkyl, Cl-4alkoxy; R and R9 are each individually
  • guanidino, Exemplifying the invention are the following compounds: (a) N-(N-Acetyl-aspartyl-glutamyl-valinyl)-3-amino-3- formylpropionic acid (b) N-(N-(l,l-Dimethylethoxycarbonyl)-aspartyl-glutamyl- valinyl)-3-amino-formylpropionic acid (c) N-(N-( 1 , 1 -Dimethy lethoxy carbony l)-aspartyl-glutamyl- valinyl)-3-amino-3-(trifluoromethylcarbonyl)propionic acid (d) N-(N-(N-(l,l-Dimethylethoxycarbonyl)anthranilyl)- aspartyl-glutamyl-valinyl)-3-amino-3-formylpropionic acid (e) N-(N-(3-(2-o
  • DIBAL diisobutyl aluminum hydride
  • EDCI l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • FAB fast atom bombardment HMPA hexamethylphosphoramide
  • TfO trifluoromethanesulfonate triflate THF tetrahydrofuran
  • TsO p-toluenesulfonate tosylate Tz 1H (or 2H)-tetrazol-5-yl
  • alkyl means linear, branched, and cyclic structures and combinations thereof, with the number of carbon atoms indicated by the prefix.
  • Alkoxy means alkoxy groups of the indicated number of carbon atoms of a straight, branched, or cyclic configuration. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, and the like.
  • Alkylthio means alkylthio groups of the indicated number of carbon atoms of a straight, branched, or cyclic configuration. Examples of alkylthio groups include methylthio, propylthio, isopropylthio, cycloheptylthio, etc. By way of illustration, the propylthio group signifies -SCH2CH2CH3. Halo includes F, Bl, Br, and I.
  • Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to diastereomers and optical isomers.
  • the present invention is meant to comprehend such possible diastereomers as well as their racemic and resolved, enantiomerically pure forms and pharmaceutically acceptable salts thereof.
  • compositions of the present invention comprise a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt, thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, ammonium, potassium, sodium, zinc and the like. Particularly preferred are the calcium, magnesium, potassium, and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, mo ⁇ holine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimemylaierine, tripropylamine, tromethamine, and the like.
  • salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.
  • acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.
  • Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.
  • the magnitude of therapeutic dose of a compound of Formula I will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound of Formula VI and its route of administration and vary upon the clinician's judgement. It will also vary according to the age, weight and response of the individual patient. An effective dosage amount of the active component can thus be determined by the clinician after a consideration of all the criteria and using is best judgement on the patient's behalf.
  • An ophthalmic preparations for ocular administration comprising 0.001-1% by weight solutions or suspensions of the compounds of Formula I in an acceptable ophthalmic formulation may be used.
  • Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention.
  • oral, parenteral and topical may be employed.
  • Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
  • compositions of the present invention comprise a compound of Formula I as an active ingredient or a pharmacetically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids.
  • compositions include compositions suitable for oral, parenteral and ocular (ophthalmic). They may be conveniently presented in unit dosage form and prepared by any of the methods well- known in the art of pharmacy. In practical use, the compounds of Formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, alcohols, oils, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case or oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amound of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into active ingredient with the carrier which constitutes one or more necessary ingredients.
  • the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
  • each tablet contains from about 1 mg to about 500 mg of the active ingredient and each cachet or capsule contains from about 1 to about 500 mg of the active ingredient.
  • a N-protected amino acid 1, with a group such as a BOC, CBZ or any other suitable nitrogen protecting group is converted to an allyl ester by a coupling reaction with l-(-3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDCI) in an inert solvent such as CH2CI2 or CHCI3 at 0°C with allyl alcohol to yield 2-
  • EDCI l-(-3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride
  • This .amino acid is N-deprotected with an acid such as HBr in MeOH in the case where the nitrogen was protected by a BOC group to yield the free amine.
  • the deprotection use is described in "Protective groups in Organic Synthesis" 2nd ed.
  • the amine is coupled as described above with _ to yield the dipeptide 4. After deprotection of the amine according to the previous conditions, it is coupled with ⁇ to yield the tripeptide £.
  • the BOC group on 5, can also be replaced by a fluorogenic substrate such as a 2-t-butyloxy- carbonylamino benzoate.
  • Deprotection of the allyl group with palladium (0) and pyrrolidine affords the acid which is coupled with n-allyloxycarbonyl-4-amino-5- benzyloxy-2-oxotetrahydrofuran 2 according to "Bio. Med. Chem. Lett.” 1992, 2, 613, to yield & after benzyl deprotection under reductive conditions.
  • R2 in 7 could be an H, CH3, CF3, OMe and SMe.
  • the cDNA encoding human poly(ADP-ribose)polymerase (clone pCD-12; GenBank accession no. M32721; NCBI gi 190266) was excised from its cloning vector by Xho I restriction digestion then ligated into Xho I-cut, CIP-treated pBluescript II SK+ (Stratagene). Following transformation into competent Escherichia coli cells, colony purification and propagation of the resulting transformed cells in liquid culture, the plasmid DNA was purified and the orientation of the PARP cDNA was determined by restriction enzyme analysis.
  • Incubation mixtures (25 ⁇ l final volume) were prepared in a buffer composed of 10 mM Hepes/KOH (pH 7.4), 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol and contained 5 ⁇ l of purified [35s]PARP, 0-10 ⁇ l of PARP cleavage activity (eg., fractions from apoptotic osteosarcoma,THP-l or other cells, or purified apopain or recombinant apopain) plus drug, where indicated, or vehicle.
  • a buffer composed of 10 mM Hepes/KOH (pH 7.4), 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol and contained 5 ⁇ l of purified [35s]PARP, 0-10 ⁇ l of PARP cleavage activity (eg., fractions from apoptotic osteosarcoma,THP-l or other cells, or
  • a fluorogenic derivative of the tetrapeptide recognized by apopain and corresponding to the Pi to P4 amino acids of the PARP cleavage site, Ac-DEVD-AMC (AMC, amino-4-methylcoumarin) was prepared as follows: i) synthesis of N-Ac-Asp(OBn)-Glu(OBn)-Val- CO2H, ii) coupling with Asp(oBn)-7-amino-4-methylcoumarin, iii) removal of benzyl groups.
  • Standard reaction mixtures 300 ⁇ l final volume, contained Ac-DEVD-AMC and purified or crude PARP-cleavage apopain/CPP32 enzyme in 100 mM Hepes/KOH (pH 7.5), 10% (w/v) sucrose, 0.1% (w/v) CHAPS, 10 mM dithiothreitol, and were incubated at 25°C. Reactions were monitored continuously in a spectrofluorometer at an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
  • FIG. 1 shows PARP cleavage activity in spontaneously apoptotic osteosarcoma cells, a, Structure of PARP and fragments resulting from proteolytic cleavage.
  • Poly(ADP-ribose) polymerase is a 113 kDa nuclear protein comprised of three functional domains: an amino-terminal DNA binding domain which contains two zinc-finger motifs that selectively recognize either single-stranded or double- stranded DNA breaks, a carboxy-terminal catalytic domain, and a central region where automodification occurs that subsequently alters DNA binding affinity37.
  • Cytosolic extracts were prepared from cultured human osteosarcoma cells (143.98.2; ATCC CRL 11226) and THP-1 cells (ATCC TIB 202) by homogenizing PBS-washed cell pellets in 10 mM Hepes/KOH (pH 7.4), 2 mM EDTA, 0.1 % (w/v) CHAPS, 5 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 10 ⁇ g/ml pepstatin A, 20 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin (at 1 x 108 cells/ml) and recovering the supernatant after successive centrifugation at 1000 xg, 10,000 xg then 100,000 xg.
  • Chicken S/M extracts were prepared from DU249 hepatoma cells38 that were committed to apoptosis by S-phase aphidicolin arrest followed by M-phase accumulation with nocodazole as described previously35.
  • the full length cDNA clone for PARP (pcD- 12)39 was excised and ligated into the Xho I site of pBluescript-II SK+ (Stratagene) then used to drive the synthesis of [35S]methionine-labelled PARP by coupled transcription (T7 polymerase)/translation (rabbit reticulocyte lysate) (Promega).
  • [35S]PARP was separated from the constituents of the transcription/translation mixture by gel filtration chromatography on a Superdex-75 FPLC column (Pharmacia; 1 x 30 cm) in 10 mM Hepes/KOH (pH 7.4), 2 mM EDTA, 0.1% (w/v) CHAPS, 5 mM dithiothreitol.
  • Reaction mixtures containing [35SJPARP (25 ⁇ l final volume in 50 mM Pipes/KOH (pH 6.5), 2 mM EDTA, 0.1% (w/v) CHAPS, 5 mM dithiothreitol) were incubated for 1 hr at 37°C in the absence (lane 1) or presence (lanes 2-6) of cytosolic extracts, including 4.5 ⁇ g protein from the cytosol fraction of non-apoptotic osteosarcoma cells (from 3 day, pre-confluent cultures) (lane 2), 4.5 ⁇ g protein from the cytosol fraction of apoptotic osteosarcoma cells (from 7 day, post- confluent cultures) (lane 3), 30 ⁇ g protein from the cytosol fraction of THP-1 cells (lane 4), 30 ⁇ g protein from a THP-1 cell cytosol fraction that was activated by pre-incubation for 60 min at 37°C) (lane 5) or 0.6 ⁇ g protein from chicken S/M extracts (
  • PARP cleavage was quantified by laser densitometry of the 24 kDa band on the resulting fluorograms. Data are the average of two independent experiments. ICE activity (open squares) was measured by the cleavage of [35S]proIL-l ⁇ essentially as described above for [35SJPARP above except at pH 7.4.
  • Figure 2 shows inhibition of PARP cleavage in apoptotic osteosarcoma cell extracts, a. Inhibition by various protease inhibitors. The cytosol fraction from apoptotic osteosarcoma cells was incubated with [35S]PARP (derived by in vitro transcription/translation) in the presence of various protease inhibitors as indicated.
  • the 24 kDa cleavage product from the resulting fluorogram is shown, b.
  • Inhibition by synthetic tetrapeptide aldehydes The cytosol fraction from apoptotic osteosarcoma cells was incubated with [35S]PARP in the presence of the indicated concentrations of the tetrapeptide aldehyde Ac-DEVD-CHO (open circles) or Ac-YVAD-CHO (solid squares) which were modeled after the P1-P4 amino acids of the PARP cleavage site and proIL-l ⁇ cleavage site, respectively.
  • the structure of Ac-DEVD-CHO is shown in the inset. METHODS, a.
  • [35SJPARP cleavage was measured as described in Fig. ⁇ b except that i) the concentration of dithiothreitol was lowered from 5 mM to 1 mM in the chromatography of the transcription/translation mixture, the cell lysis buffer and the [35s]PARP cleavage incubation buffer, and ii) the cell lysis buffer did not contain protease inhibitors.
  • Incubation mixtures containing 10 ⁇ g protein from the cytosol fraction of apoptotic osteosarcoma cells (from 7 day, post-confluent cultures) were pre-incubated for 20 min at 37°C with the indicated protease inhibitor before the addition of [35S]PARP.
  • Incubation mixtures contained 100 ⁇ M 4-amidino-phenyl-methane-sulfonyl fluoride (pAPMSF; lane 3), 2 ⁇ g/ml aprotinin Qane 4), 100 ⁇ M elastinal (lane 5), 1 mM phenylmethylsulfonylfluoride (PMSF, lane 6), 100 ⁇ M L-l- chloro-3-[4-tosylamido]-7-amino-2-heptanone (TLCK, lane 7), 100 ⁇ M L-l-chloro-3-[4-tosylamido]-4-phenyl-2-butanone (TPCK, lane 8), 1 mg ml soybean trypsin inhibitor (SB-TI, lane 9), 10 ⁇ M amastatin (lane 10), 10 ⁇ M bestatin (lane 11), 50 ⁇ M diprotin A (lane 12), 8.5 ⁇ M phosphoramidon (lane 13), 1 ⁇ M
  • Incubation mixtures containing 10 ⁇ g protein from the cytosol fraction of apoptotic osteosarcoma cells were pre-incubated for 20 min at 37 °C with the indicated concentrations of Ac-YVAD-CHO (solid squares) or Ac-DEVD-CHO (open circles) before the addition of [35S]PARP.
  • the incubation was continued at 37°C for 60 min then the samples were resolved on 10% SDS/polyacrylamide gels. Cleavage products were visualized by fluorography of the resulting dried gel and the band corresponding to the 24 kDa cleavage product was quantified by laser densitometry. Data are expressed as the percentage of the control to which no inhibitor was added and are the average of two independent experiments.
  • Figure 3 shows purification of the PARP cleavage protease from THP-1 cells, a. DEAE anion-exchange chromatography. b. Structure of biotinylated tetrapeptide-aldehyde affinity ligands. c.
  • the cytosol fraction from cultured THP-1 cells was isolated, dialysed and concentrated as described previously41 then applied to a DEAE-5PW HPLC column (TosoHaas, 5.5 x 20 cm; 3-5 gm protein from 1.4 x 10l 1 cells) that had been pre-equilibrated at 4°C in 20 mM Tris/HCl (pH 7.8), 10% (w/v) sucrose, 0.1% (w/v) CHAPS, 2 mM dithiothreitol.
  • Proteins were eluted with a linear gradient of 0.4 M NaCl, 240 mM Tris/HCl (pH 7.8), 10% (w/v) sucrose, 0.1% (w/v) CHAPS, 2 mM dithiothreitol. Fractions corresponding to approximately 90 to 120 mM NaCl, which immediately followed those containing ICE activity, were pooled and the pools from 25 DEAE chromatography runs were combined (1.6 gm protein, from 3.5 x 1012 THP-1 cells) and re-run under identical conditions. The PARP cleavage activity was assayed in each fraction as described in Fig lb and quantified by laser densitometry as described in Fig 2b.
  • Biotin-DEVD-CHO and Biotin-[X]-DEVD-CHO differ by the presence of a 0.9 nm spacer arm (indicated by the square brackets) which is present in Biotin-[X]-DEVD-CHO but absent in Biotin-DEVD- CHO.
  • ligands were prepared by: i) synthesis of t-Boc-Asp(OBn)- Glu(OBn)-Val-Asp-CHO protected as the benzylated lactol at the aldehyde, ii) removal of the t-Boc group, iii) acylation of the free .amine with biotin (for Biotin-DEVD-CHO) or biotinamidocaproic acid (for Biotin-[X]-DEVD-CHO) using EDCI and HOBt.
  • biotin for Biotin-DEVD-CHO
  • biotinamidocaproic acid for Biotin-[X]-DEVD-CHO
  • fraction 114 The fraction from DE.AE chromatography corresponding to the peak of P.ARP cleavage activity (fraction 114; 2.5 ml containing 3 mg protein) was incubated with 20 nmol of Biotin-[X]- DEVD-CHO in a total volume of 10 ml of 50 mM PIPES/KOH (pH 6.8), 2 mM EDTA, 0.1 % (w/v) CHAPS, 5 mM dithiothreitol for 30 min at room temperature.
  • the enzyme was eluted by perfusing the column with 2 mM D-biotin in the same buffer and allowing it to stand for several hours before recovering the purified PARP cleavage enzyme.
  • An identical affinity chromatography run using Biotin-DEVD-CHO yielded comparable results.
  • Samples were resolved on 14% SDS/polyacrylamide gels and protein bands were visualized by silver staining.
  • Samples contained 9 ⁇ g protein from a THP-1 cell cytosol fraction (lane 1), 6 ⁇ g protein from DEAE fraction 114 before (lane 2) and after (lane 3) Biotin-[X]-DEVD-CHO affimty chromatography, and 0.1 ⁇ g protein from the eluent of Biotin-DEVD- CHO (lane 5) and Biotin-[X]-DEVD-CHO (lane 6) affinity columns.
  • Figure 4 shows the structure of PARP cleavage protease; apopain, which is processed from the inactive proenzyme CPP32 a.
  • Electrospray mass spectroscopy analysis of 17 kDa (left) and 12 kDa (right) subunits of the purified PARP cleavage protease are shown.
  • b. Primary structure of apopain/CPP32. The deduced amino acid sequence from the CPP32B cDNA clone25 is shown. Hatched bars indicate the aimiic erminal sequences determined for the purified enzyme subunits.
  • Arrowheads mark the Asp28-Ser29 and Aspl75-Serl76 cleavage sites which yield the pi 7 and pl2 subunits from the CPP32 proenzyme.
  • Numbering corresponds to the residue position within human ICE. Regions implicated in substrate binding to human ICE, based on the X-ray crystal structure33,34 > are shown: solid circles, catalytic; open circles, binding pocket for carboxylate of Pi Asp; triangles, proximity ( ⁇ 0.4 nm) to P2-P4 residues. Arrowheads indicate known proenzyme cleavage sites for ICE and CPP32.
  • Fig 3c Approximately 100 pmol of the purified PARP cleavage enzyme described in Fig 3c was resolved on a 14% SDS/polyacrylamide and transferred to a polyvinylidenedifluoride membrane by electroblotting. Regions of the membrane containing the individual pi 7 and pl2 subunits were excised and sequenced by conventional Edman degradation using a continuous- flow reactor (Sheldon Biotechnology Centre, Montreal Canada). Hatched bars represent the resulting ain o-terminal sequences which perfectly corresponded to the deduced amino acid sequence of CPP32B.
  • hICE re l-II and hlCErel-III are human ICE-related cysteine protease II and III, respectively;
  • mICE, rICE and hICE are murine, rat and human ICE (interleukin-l ⁇ converting enzyme), respectively;
  • mNedd2 and hICH-1 are the murine and human forms of Nedd2/ICH-lL» respectively;
  • cbCED-3, cvCED-3 and ceCED-3 are Caenorhabditis briggsae, v lgaris and elegans CED-3 (cell-death-abnormal ced-3 gene product), respectively;
  • hCPP32 is human CPP32 ⁇ .
  • Figure 5 shows the kinetic analysis of apopain and a potent inhibitor using a fluorogenic substrate
  • a Determination of Km for Ac-DEVD-AMC (structure in insert)
  • b Kinetics of inhibition of CPP32 by the peptide aldehyde Ac-DEVD-CHO.
  • c Comparison of PARP cleavage activity and inhibition by Ac-DEVD-CHO in THP-1 cell, osteosarcoma cell and chicken S/M extracts. METHODS, a.
  • Ac-DEVD-AMC (inset) (AMC, amino-4- methylcoumarin) was prepared as follows: i) synthesis of N-Ac- Asp(OBn)-Glu(OBn)-Val-C ⁇ 2H, ii) coupling with Asp(oBn)-7-amino- 4-methylcoumarin, iii) removal of benzyl groups.
  • the Km for cleavage of the synthetic fluorogenic tetrapeptide Ac-DEVD-AMC and the kon and Ki values for the tetrapeptide aldehyde inhibitor Ac- DEVD-CHO were determined as described above for panels a and b, respectively.
  • Figure 6 shows in vitro apoptosis and selective inhibition by Ac-DEVD-CHO or by depletion of apopain-mediated PARP cleavage activity
  • a Cytosols from progressively apoptotic osteosarcoma cells confer apoptotic changes upon healthy nuclei from non-apoptotic cells.
  • the cytosol fraction from osteosarcoma cells at various stages of apoptotic death were incubated with isolated nuclei from non-apoptotic osteosarcoma cells and mo ⁇ hological changes were assessed by fluorescent microscopy after staining with Hoechst 33342.
  • b Cytosols from progressively apoptotic osteosarcoma cells confer apoptotic changes upon healthy nuclei from non-apoptotic cells.
  • the cytosol fraction from osteosarcoma cells at various stages of apoptotic death were incubated with isolated nuclei from non-apoptotic osteosarcoma cells and mo ⁇ hological changes were
  • Col. 9 Pro-apoptotic osteosarcoma cell cytosols were depleted of PARP cleavage activity (columns 11-15) then incubated with healthy nuclei from non-apoptotic osteosarcoma cells in the presence of varying amounts of purified apopain (col. 12-14) or purified ICE (col. 15).
  • RESULTS Osteosarcoma cells at various stages of apoptosis and cytosolic extracts from them were prepared as described for Fig. 1. Nuclei were isolated from non-apoptotic (day 3) cells essentially as described before36 except that the nuclear isolation buffer was 10 mM Pipes/KOH (pH 7.4), 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 10 ⁇ M cytochalasin B, 1 mM phenylmethylsulfonyl fluoride, 10 ⁇ g/ml pepstatin A, 20 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin. a.
  • the isolated nuclei from 2 x 106 day-3 cells were combined with 25 ⁇ l of the cytosol fraction (2.5 x 106 cell equivalents) from cells maintained for the indicated times in culture then incubated in 100 ⁇ l (final volume) of a mixture containing 10 mM Hepes/KOH (pH 7.0), 50 mM NaCl, 2 mM MgCl2, 0.1 mM CaCl2, 40 mM ⁇ -glycerophosphate, 1 mM dithiothreitol, 2 mM ATP, 10 mM creatine phosphate and 50 ⁇ g/ml creatine kinase.
  • nuclear chromatin was stained with 5 ⁇ g/ml Hoechst 33342 and examine by fluorescent microscopy (excitation wavelength 330 nm; emission wavelength 450 nm). Nuclei having brightly fluorescent, condensed and fragmented chromatin were scored as apoptotic whereas non-apoptotic nuclei were identified by weakly fluorescent, uniform chromatin staining. For each condition, a minimum of 250 nuclei in 5 separate fields were scored. Data are the average of two independent experiments, b. Col.
  • cleavage (between As ⁇ 216 and Gly 17) separates the amino- terminal DNA-nick sensor of PARP from its carboxy-te ⁇ ninal catalytic domain (Fig. la).
  • [35S]PARP was generated as a substrate by in vitro transcription/translation of a fiill-length human PARP cDNA clone and was then combined with various cell extracts (Fig. lb).
  • a human osteosarcoma cell line with a propensity for spontaneous apoptotic death contained substantial PARP cleavage activity that was markedly higher in extracts from apoptotic cells versus non-apoptotic cells (lane 2 vs 3).
  • Osteosarcoma cells are a good model system for studying the events that occur during apoptosis. Upon reaching confluence in culture they undergo the mo ⁇ hological and biochemical changes characteristic of apoptotic death, including cell shrinkage, membrane blebbing, chromatin condensation and fragmentation (not shown) as well as internucleosomal DNA cleavage (Fig. lc).
  • the PARP cleavage activity measured in cell extracts was elevated > 10-fold (Fig. Id).
  • PARP cleavage activity was also measurable in cytoplasmic extracts of THP-1 cells, the human monocytic leukemia cell line from which ICE was originally purified, particularly after pre-incubation of the extracts at 37°C (Fig. lb, lane 4 vs 5). This suggests that the PARP cleavage enzyme requires activation of a latent form as has been described for ICE in this cell line30.
  • PARP cleavage in apoptotic osteosarcoma cell extracts and activated THP-1 cell extracts was comparable to that in apoptotic chicken S/M extracts (lane 6) where this proteolytic activity was originally identified 19.
  • a tetrapeptide aldehyde containing the P1-P4 amino acid sequence of the PARP cleavage site (DEVD216-G217) was therefore synthesized and found to be a potent inhibitor of PARP breakdown (Ac-DEVD-CHO; Fig. 2b inset).
  • Ac-DEVD-CHO iiihibited the PARP cleavage activity in apoptotic osteosarcoma cell extracts with an IC50 value of 0.2 nM.
  • the corresponding carboxylic acid (Ac-DEVD-COOH) and the tetrapeptide aldehyde containing the proIL-l ⁇ recognition sequence for ICE (Ac-YVAD-CHO) had IC50 values >10 ⁇ M (Figs. 2a & 2b).
  • An identical inhibitor profile was found for the PARP cleavage activity in activated THP-1 cell extracts and in apoptotic chicken S/M extracts (not shown).
  • the cowpox- virus se ⁇ in CrmA (the cytokine-response- modifier A (crmA) gene product), which is a potent inhibitor of ICE31 (Ki ⁇ 4 pM), had no inhibitory effect on PARP cleavage when tested up to 0.6 ⁇ M (not shown).
  • PARP cleavage is therefore mediated by an E- 64-insensitive cysteine protease that can be inhibited by low concentrations of the tetrapeptide aldehyde Ac-DEVD-CHO but not by high levels of potent inhibitors of ICE.
  • biotinylated derivatives of the Ac- DEVD-CHO tetrapeptide aldehyde inhibitor were synthesized as affinity ligands for the PARP cleavage enzyme (Fig. 3b).
  • Biotinylated affinity ligands were used because they could be pre-incubated with the enzyme in order to overcome slow ligand binding (see below) by allowing full equilibrium to occur prior to harvesting.
  • Both biotinylated tetrapeptide aldehydes had IC50 values for inhibition of the PARP cleavage enzyme that were comparable to that of the non-biotinylated parent compound (0.2 nM; not shown).
  • the DEAE-chromatography fraction at the peak of PARP cleavage activity was incubated with the biotinylated tetrapeptide aldehydes then harvested with streptavidin-agarose. After extensive washing, the purified PARP cleavage enzyme was eluted from the column with 2 mM biotin. SDS/polyacrylamide gel electrophoresis of the resulting samples indicated that the purified PARP cleavage enzyme was composed of two major polypeptides of approximately 17 and 12 kDa (Fig. 3c).
  • PARP cleavage enzyme indicated that the mass of the larger polypeptide was 16,617.1 ⁇ 3.1 and the smaller polypeptide was 11,896.2 ⁇ 1.2 (Fig. 4a).
  • Amino-terminal sequence determination and tryptic maps of the purified apopain enzyme identified it as a protolytic product of the inactive CPP32 proenzyme, a member of the ICE CED-3 family of cysteine proteases of unknown function that was recently cloned from Jurkat cells25.
  • Cloned CPP32 was originally identified as two isoforms (CPP32a and CPP32B) which differ by a single conservative amino acid substitution (Aspl90 V s Glul90 for CPP32a and CPP32B, respectively).
  • a continuous fluorometric assay for apopain was developed with the substrate Ac-DEVD-AMC (AMC, amino-4-methylcoumarin).
  • the design of this substrate was based on the tetrapeptide- AMC motif that has been used successfully with ICE7, except using the PARP cleavage site P1-P4 tetrapeptide (Fig. 5 A inset).
  • Peptide aldehydes are potent, reversible inhibitors of cysteine proteases that undergo nucleophilic addition of the catalytic cysteine to form a thiohemiacetal.
  • the potency of aldehyde inhibitors was originally attributed to their ability to mimic the transition state in amide bond hydrolysis35, the recently determined crystal structure of ICE with the tetrapeptide aldehyde Ac-YVAD-CHO clearly shows this inhibitor bound in a non-transition-state conformation, with the oxyanion of the thiohemiacetal being stabilized by the active site histidine33.
  • the tetrapeptide aldehyde containing the appropriate recognition sequence for apopain, Ac-DEVD-CHO is a potent, competitive inhibitor of this enzyme. It is slow binding, as shown by the time-dependent approach to equilibrium observed when enzyme was added to reaction mixtures containing inhibitor (50 nM) and 1 x Km substrate (Fig. 5b).
  • the crystal structure of active ICE has indicated that the two key amino acids that interact with the P4 Tyr of proIL-l ⁇ are His342 and Pro343 which are replaced by Asn and Ser, respectively, in both apopain/CPP32 and CED-3 (Fig. 4e). These latter residues in apopain/CPP32 would be better able to form the hydrogen bonds necessary to interact with the carboxylate side chain of the P4 Asp of PARP.
  • the enzymes also clearly have different macromolecular substrate specificities: purified ICE was unable to cleave PARP and purified apopain did not cleave proIL-l ⁇ at either the FEAD27-G28 or DEVD216-G 17 cleavage sites (a 5000-fold excess of each enzyme was tested; not shown).
  • the enzymes are also distinguished by their behaviour with the cowpox se ⁇ in, CrmA, which shows a more than 10,000-fold preference for ICE.
  • Apoptotic events can be re-constituted in vitro.
  • the isolated nuclei from healthy cells undergo the mo ⁇ hological changes that are characteristic of apoptosis (eg. chromatin condensation, fragmentation and margination as well as internucleosomal DNA cleavage) when they are incubated with the cytosol fraction from apoptotic cells36. Since the most potent and selective inhibitor of apopain-mediated PARP cleavage (Ac-DEVD-CHO) was membrane impermeable and hence inactive in intact cells, this system was established with human cells and used to study the effects of apopain inhibition or depletion on apoptosis in vitro.
  • Cytosols from non- apoptotic osteosarcoma cells had little effect on nuclear mo ⁇ hology whereas those from progressively apoptotic cells were capable of inducing apoptosis-like changes in the recipient nuclei (Fig. 6a).
  • the degree of apoptotic mo ⁇ hology conferred upon the otherwise healthy nuclei coincided with the degree of apoptosis occurring in the cells from which the cytosols were extracted (cf. Fig. lc) as well as the level of PARP cleavage activity (cf. Fig. Id).
  • Vectors containing the apopain encoding DNA sequence are used to drive the translation of the apopain polypeptide in rabbit reticulocyte lysates, mammalian host cells, and in baculovirus infected insect cells.
  • the experimental procedures are essentially those outlined in the manufacturers' instructions.
  • SK+:apopain plasmid DNA (with apopain in the T7 orientation) is linearized by Bam HI digestion downstream of the apopain insert.
  • the linearized plasmid is purified and used as a template for run-off transcription using T7 RNA polymerase in the presence of m7G(5')ppp(5')G.
  • the resulting capped apopain transcripts are purified by lithium chloride precipitation and used to drive the translation of apopain in nuclease-pretreated rabbit reticulocyte lysate in the presence of L-[35s]methionine.
  • apopain protein is expressed in mammalian host cells following transfection with either pcDNA I/Amp:apopain (under control of the CMV promoter) or pSZ9016-l:apo ⁇ ain (under control of the HTV LTR promoter).
  • pSZ9016-l:apopain cells are co-transfected with the TAT expressing plasmid pSZ90161:TAT.
  • COS-7 cells are transfected using either DEAE-dextran or lipofection with Lipofectamine (BRL).
  • apopain - containing baculovirus transfer vector pVL1393:T7 apopain HA is used to produce recombinant baculovirus (Autographa californica) by in vivo homologous recombination.
  • Epitope tagged apopain is then expressed in Sf9 (Spodoptera frugiperda) insect cells grown in suspension culture following infection with the apopain - containing recombinant baculovirus.
  • EXAMPLE S Cloning Of Of Apoptain For Expression Of The Apoptain Polypeptide In Other Host Cell Systems a) Cloning of apopain cDNA into a bacterial expression vector.
  • Recombinant apopain is produced in a bacterium such as E.coli following the insertion of the optimal apopain cDNA sequence into expression vectors designed to direct the expression of heterologous proteins.
  • These vectors are constructed such that recombinant apopain is synthesized alone or as a fusion protein for subsequent manipulation. Expression may be controlled such that recombinant apopain is recovered as a soluble protein or within insoluble inclusion bodies.
  • Vectors such as pBR322, pSKF, pUR, pATH, pGEX, ⁇ T7-5, pT7-6, pT7-7, pET, pEBI (IBI), pSP6 T7-19 (Gibco/BRL), pBluescript II (Stratagene), pTZ18R, pTZ19R (USB), pSE420 (Invitrogen) or the like are suitable for these purposes.
  • apopain cDNA into a veast expression vector
  • Recombinant apopain is produced in a yeast such as Saccharomyces cerevisiae following the insertion of the optimal apopain cDNA cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins.
  • vectors such as EmBLyex4 or the like are ligated to the apopain cistron [Rinas, U. et al., Biotechnology 8: 543-545 (1990); Horowitz B. et al., J. Biol. Chem. 265: 4189-4192 (1989)].
  • the apopain cistron is ligated into yeast expression vectors which fuse a secretion signal (a yeast or mammalian peptide) to the amino terminus of the apopain protein [Jacobson, M. A., Gene 85: 511-516 (1989); Riett L. and Bellon N. Biochem. 28: 2941- 2949 (1989)].
  • apopain cDNA is produced in mammalian host cells, such as HeLa S3 cells, after infection with vaccinia virus containing the apopain cDNA sequence.
  • the apopain cDNA is first ligated into a transfer vector, such as pSCl 1, pTKgptFls, pMJ601 or other suitable vector, then transferred to vaccinia virus by homologous recombination. After plaque purification and virus amplification, apopai vaccinia virus is used to infect mammalian host cells and produce recombinant apopain protein.
  • a transfer vector such as pSCl 1, pTKgptFls, pMJ601 or other suitable vector
  • Recombinant apopain is produced by a) transforming a host cell with DNA encoding apopain protein to produce a recombinant host cell; b) culturing the recombinant host cell under conditions which allow the production of apopain; and c) recovering the apopain.
  • the recombinant apopain is purified and characterized by standard methods.
  • EXAMPLE 10 Compounds that modulate apopain activity may be detected by a variety of methods.
  • a method of identifying compounds that affect apopain comprises:
  • Compounds that modulate apopain activity may be formulated into pharmaceutical compositions. Such pharmaceutical compositions may be useful for treating diseases or conditions that are characterized by altered apopain activity. Examples of diseases wherein the apopain activity is increased include immune deficiency syndromes, pathogenic infections, cardiovascular and neurological injury, alopecia, aging, Parkinson's disease and Alzheimers disease. For these diseases, therapeutic treatment comprises treatment with compounds that decrease the apopain activity. Examples of diseases wherein the apopain activity is decreased include autoimmune diseases, leukemias, lymphomas and other cancers. For these diseases, therapeutic treatment comprises treatment with compounds that increase apopain activity.
  • Step 3 N-Ac-Asp-rBz,-GlufBzVVal-Asp(di-bz, To a mixture of the amine (0.043 g, 0.13 mmol) of Step 2,
  • Example 11 acid (0.074 g, 0.12 mmol) of Step 6, Example 12 and HOBt (0.021 g, 0.15 mmol) in 0.5 mL of CHCI3 at 0°C was added EDCI (0.034 g, 0.18 mmol). Similar workup to that in Step 1 of Example 11 afforded 0.105 g (94%) of the title compound after chromatography on silica gel (eluted with 5% MeOH in CHCI3).
  • Step 6 N- Acety l-L-( ⁇ -benzylasparty 1 )-L-(g-benzy lgl utamy 1 )-L- valin ⁇
  • Step 7 N-(N-Acetyl-L-aspartyl- ⁇ -benzyl ester-L-glutamyl-g- benzyl ester-L-valine)-4-amino-5-benzyloxy-2- oxotetrahvdrofuran
  • N-acetyl-L-( ⁇ -benzylaspartyl)-L-(g- benzylglutamyl)-L-valine 608 mg, 1.04 mmol
  • N- allyloxycarbonyl-4-amino-5-benzyloxy-2-oxotetrahydrofuran 335 mg, 1.15 mmol
  • PdCl2(PPh3)2 43 mg, .060 mmol
  • Bu3SnH 310 mL, 1.15 mmol
  • Step 8 N-(N-Acetyl-L-aspartyl-L-glutamyl-L-valinyl)-3-amino-3- formylpropionic acid
  • step 7 To as suspension of the product of step 7 (51 mg, 66 mmol) in 5 mL MeOH was added 50 mg of Pd(OH)2(20% on carbon). The mixture was stirred under an atmosphere of H2 for 24 h. The catalyst was filtered on celite, washed with 10 mL MeOH. The MeOH extract was concentrated and flash chromatographed on silica gel (CHCl3/MeOH 5%) to afford, after lyophilizing the residue from 5 mL H2 ⁇ &10 mL AcOH, 17 mg (47%) of the title compound.

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L'invention concerne un composé de la formule (I): R?1COAA1AA2AA3¿NHX. Ces composés sont des inhibiteurs de iapopaïne, une enzyme impliquée dans le processus d'apoptose. Ces composés sont utiles en tant qu'outils de recherche, ainsi que pour le traitement de différents troubles, où une diminution de l'apoptose serait bénéfice, en particulier les syndromes d'immunooléficience (en particulier le SIDA), le diabète type I, les infections par des pathogènes, les atteintes cardiovasculaires et neurologiques, l'alopécie, le vieillissement, la maladie de Parkinson et la maladie d'Alzheimer.
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WO1999050230A1 (fr) * 1998-03-31 1999-10-07 Vertex Pharmaceuticals Incorporated Inhibiteurs de serine protease, particulierement de la protease ns3 du virus de l'hepatite c
US6303374B1 (en) 2000-01-18 2001-10-16 Isis Pharmaceuticals Inc. Antisense modulation of caspase 3 expression
EP1165490A4 (fr) * 1999-03-16 2002-09-18 Cytovia Inc Inhibiteurs de caspases 2-aminobenzamidiques substitues et leur utilisation
US6531474B1 (en) 1998-03-19 2003-03-11 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
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US6984718B2 (en) 1998-07-21 2006-01-10 Cytovia, Inc. Fluorescence dyes and their applications for whole-cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof
US7153822B2 (en) 2002-01-29 2006-12-26 Wyeth Compositions and methods for modulating connexin hemichannels
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WO1998022098A3 (fr) * 1996-11-20 1998-07-09 Qlt Phototherapeutics Inc Inhibiteurs de cpp32 destines a reguler l'apoptose
US7270801B2 (en) 1997-10-10 2007-09-18 Cytovia, Inc. Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for caspases and other enzymes and the use thereof
EP1026988A4 (fr) * 1997-10-10 2005-03-30 Cytovia Inc Nouvelles molecules reporters fluorescentes, applications de ces molecules et dosages des caspases
US6531474B1 (en) 1998-03-19 2003-03-11 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
EP2261235A2 (fr) 1998-03-19 2010-12-15 Vertex Pharmaceuticals Incorporated Inhibiteurs de caspase
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US6608067B1 (en) 1998-03-31 2003-08-19 Vertex Pharmaceuticals Incorporated Inhibitors of serine proteases, particularly HCV NS3 protease
WO1999050230A1 (fr) * 1998-03-31 1999-10-07 Vertex Pharmaceuticals Incorporated Inhibiteurs de serine protease, particulierement de la protease ns3 du virus de l'hepatite c
US6984718B2 (en) 1998-07-21 2006-01-10 Cytovia, Inc. Fluorescence dyes and their applications for whole-cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof
US6620782B1 (en) 1999-03-16 2003-09-16 Cytovia, Inc. Substituted 2-aminobenzamide caspase inhibitors and the use thereof
EP1165490A4 (fr) * 1999-03-16 2002-09-18 Cytovia Inc Inhibiteurs de caspases 2-aminobenzamidiques substitues et leur utilisation
US6716818B2 (en) 1999-04-09 2004-04-06 Cytovia, Inc. Caspase inhibitors and the use thereof
US6303374B1 (en) 2000-01-18 2001-10-16 Isis Pharmaceuticals Inc. Antisense modulation of caspase 3 expression
US7417029B2 (en) 2000-05-19 2008-08-26 Vertex Pharmaceuticals Incorporated Prodrug of an ice inhibitor
EP2270005A1 (fr) 2000-05-19 2011-01-05 Vertex Pharmceuticals Incorporated Promédicament inhibiteur d'ECI
US8022041B2 (en) 2000-05-19 2011-09-20 Vertex Pharmaceuticals Incorporated Prodrug of an ICE inhibitor
US8329662B2 (en) 2000-05-19 2012-12-11 Vertexd Pharmaceuticals Incorporated Prodrug of an ICE inhibitor
US9156880B2 (en) 2000-05-19 2015-10-13 Vertex Pharmaceuticals Incorporated Prodrug of an ice inhibitor
US9487555B2 (en) 2000-05-19 2016-11-08 Vertex Pharmaceuticals Incorporated Prodrug of an ice inhibitor
US9994613B2 (en) 2000-05-19 2018-06-12 Vertex Pharmaceuticals Incorporated Prodrug of an ICE inhibitor
US7153822B2 (en) 2002-01-29 2006-12-26 Wyeth Compositions and methods for modulating connexin hemichannels
US7531570B2 (en) 2004-05-27 2009-05-12 Vertex Pharmaceuticals Incorporated Treatment of diseases using ICE inhibitors
US9116157B2 (en) 2010-11-05 2015-08-25 Brandeis University Ice-cleaved alpha-synuclein as a biomarker
US11021514B2 (en) 2016-06-01 2021-06-01 Athira Pharma, Inc. Compounds

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